EXCEED THE SPACE PROVIDED. The long-range goal of this project is to define the interactions in the transcription complex that regulate pausing and termination by RNA polymerase. Nascent RNA hairpins are important regulatory signals in bacteria, where pausing and termination are major components of genetic regulatory mechanisms. Pausing and premature termination also affect expression of genes in mammalian cells and viruses, notably genes involved in the development of cancer and in growth of the AIDS virus, HIV-1. In both bacteria and eukaryotes, specialized regulatory proteins modify the transcription complex to make it resistant to pausing and termination. Although significant progress has been made in understanding pausing, termination, and the regulatory proteins that control these events, the detailed mechanisms of nucleotide addition and pausing by RNA polymerase remain uncertain. Different models for pausing are currently being evaluated, involving either binding of an allosteric nucleoside triphosphate or movements of particular parts of RNA polymerase called the bridge helix and the trigger loop. Pausing and termination by E. coli RNA polymerase and their regulation by the NusA, NusG, and RfaH proteins, and pausing by human RNA polymerase II have been developed as model systems with which to test these mechanisms and how regulators affect them. A combination of biochemical, genetic, and biophysical approaches will be used to test the bridge-helix, trigger-loop, and allosteric nucleoside-triphosphate models of pausing, and to characterize the how regulators like NusA, NusG, and RfaH control RNA polymerse. Specific aims will be to (/) characterize interactions of RNA polymerase's flap-tip helix with RNA,NusA, and a70, and test how these interactions affect catalysis in the active site; (//) determine the location of the RNA 3' end in paused and nonpaused transcription elongation complexes; (Hi)determine the kinetic mechanisms of elongation, pausing, and termination; (iv)map interactions between RNA polymerase and pause and terminator hairpins; and (v) determine the sites at which RfaH and NusG interact with RNA polymerase and the mechanisms by which they regulate transcript elongation.